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Questions...agree greendisagree red

• T-DNA is encoded on the Ti-plasmid of
  Agrobacterium tumefaciens
• T-DNA inserts in random places in the genome
• In order for a transposable element to jump, a
  second element encoding a transposase needs to be
  present.
• Transposable elements insert in random places in
  the genome
• The ultimate proof that you have clone the gene
  you were after is showing that different mutant
  alleles of the cloned gene all result in similar
  mutant phenotypes.
• A co-segregating fragment is a DNA fragment with
  a known insertion that is present in the genome
  of all individuals that carry a mutant allele.
• Plasmid rescue is only possible with transformed
  (i.e. transgenic) plants.

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• Summary of T-DNA and transposon-tagging in
  targeted cloning
• There is a mutant of interest. We would like to
  know what gene is mutated, but we have no clue
  what that gene might be.
• We use a known sequence (T-DNA or transposon)
  that inserts by chance in the wild-type gene and
  results in a new mutation that is allelic to the
  original mutation. Any mutants we find are selfed
  and backcrossed.
• We use the fact that we know the sequence of the
  mutating agent to locate the gene of interest.
  This typically involves identifying a
  co-segregating fragment on a Southern blot that
  contains DNA from mutants and wild-type siblings.
  Ideally, the co-segregating fragment, which is
  always present in the mutants and always absent
  in the wild types, contains the mutating agent
  and is flanked by the DNA of the gene of
  interest.
• We clone the co-segregating fragment via IPCR,
  plasmid rescue or a library and sequence the
  flanking DNA. This is then expected to be part
  of the gene of interest.
• To prove that this DNA is really the gene of
  interest, we can either
• Transform a mutant with a wild-type copy of the
  gene. This is expected to result in wild-type
  transformants.
• Identify several independent, yet allelic
  mutations and show that in each case there is a
  Dna and/or RNA polymorphism associated with the
  mutant phenotype. DNA polymorphisms may show
  insertions or deletions, and RNA polymorphisms
  may show reduction in gene expression or the
  appearance of mRNAs of different sizes.

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An overview of gene cloning strategies
Is the identity of the gene known?
Is T-DNA tagging possible?
Is there a mutant in which the gene is deficient?
Has the genome been sequenced?
Clone via T-DNA tagging
Is transposon-tagging possible?
Has the gene been cloned from a different species?
Is there partial amino acid sequence available?
Clone via transposon tagging
Are there BAC or YAC clones available?
Can the gene be mapped?
Is this a closely related species?
Map-based cloning
Does the gene encode a protein that can be
assayed?
Can the protein be purified?
Are there candidate genes in the databases?
Screen a genomic or cDNA library with a
heterologous probe
RT-PCR with degenerate primers
Obtain cDNA, check expression patterns,
functional assays..
Postpone or abandon project
LD-PCR on genomic or cDNA
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Xu et al. - Glossy8
Is the identity of the gene known?
Is T-DNA tagging possible?
Is there a mutant in which the gene is deficient?
Has the genome been sequenced?
Clone via T-DNA tagging
Is transposon-tagging possible?
Has the gene been cloned from a different species?
Is there partial amino acid sequence available?
Clone via transposon tagging
Are there BAC or YAC clones available?
Can the gene be mapped?
Is this a closely related species?
Map-based cloning
Does the gene encode a protein that can be
assayed?
Can the protein be purified?
Are there candidate genes in the databases?
Screen a genomic or cDNA library with a
heterologous probe
RT-PCR with degenerate primers
Obtain cDNA, check expression patterns,
functional assays..
Postpone or abandon project
LD-PCR on genomic or cDNA
5
Meyer et al. - F5H
Is the identity of the gene known?
Is T-DNA tagging possible?
Is there a mutant in which the gene is deficient?
Has the genome been sequenced?
Clone via T-DNA tagging
Is transposon-tagging possible?
Has the gene been cloned from a different species?
Is there partial amino acid sequence available?
Clone via transposon tagging
Are there BAC or YAC clones available?
Can the gene be mapped?
Is this a closely related species?
Map-based cloning
Does the gene encode a protein that can be
assayed?
Can the protein be purified?
Are there candidate genes in the databases?
Screen a genomic or cDNA library with a
heterologous probe
RT-PCR with degenerate primers
Obtain cDNA, check expression patterns,
functional assays..
Postpone or abandon project
LD-PCR on genomic or cDNA
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Franke et al. REF8
Is the identity of the gene known?
Is T-DNA tagging possible?
Is there a mutant in which the gene is deficient?
Has the genome been sequenced?
Clone via T-DNA tagging
Is transposon-tagging possible?
Has the gene been cloned from a different species?
Is there partial amino acid sequence available?
Clone via transposon tagging
Are there BAC or YAC clones available?
Can the gene be mapped?
Is this a closely related species?
Map-based cloning
Does the gene encode a protein that can be
assayed?
Can the protein be purified?
Are there candidate genes in the databases?
Screen a genomic or cDNA library with a
heterologous probe
RT-PCR with degenerate primers
Obtain cDNA, check expression patterns,
functional assays..
Postpone or abandon project
LD-PCR on genomic or cDNA
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Cloning strategies II the random approach

• This approach is used when we are interested in
  finding a number of genes associated with a
  specific biological process.
• Typically there is a mutant phenotype that can be
  identified through screening
• A similar mutant phenotype can be caused by
  mutations in different genes
• This tends to work well for genes that are part
  of a biochemical pathway with a defined end
  product.
• epicuticular waxes (glossy mutants in maize)
• lignin (ref mutants in Arabidopsis brown midrib
  mutants in maize)
• A typical process works as follows
• Mutagenesis
• EMS or DES (chemical mutagenesis) typically
  point mutations
• Radiation-induced mutants typically (small)
  deletions
• T-DNA (insertions)
• Transposons (insertions)
• Self pollinations
• This is done to reveal the phenotype when
  mutations are recessive
• 3) Screening for mutants

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• 4) Allelism-tests
• In order to find out how many different loci are
  involved, the mutants that have been identified
  are crosses with each other.
• Allelic mutations lead to the mutant phenotype in
  the progeny.
• Cloning
• Depending on the nature of the mutagenesis the
  cloning process can begin
• Transposon-induced mutations can be cloned via
  transposon-tagging
• T-DNA induced mutants can be cloned via T-DNA
  tagging
• EMS or DES mutants can be mapped and cloned via
  map-based cloning OR additional mutant alleles
  can be created via T-DNA or transposon-tagging.
• If phenotypic data can be acquired, it may also
  be possible to use PCR based approaches this is
  referred to as the candidate-gene approach
  (Pflieger et al., 2001)
• Advantages of the random approach is that many
  (all) genes involved in a process are uncovered
  in a screening process that requires relatively
  little extra effort compared to the targeted
  approach.
• A disadvantage is that it can take some effort
  sorting out how many loci are affected.
  Depending on the mutagenesis method the cloning
  may require a targeted approach after all.

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The candidate gene approach (Pflieger et al.,
2001)
This approach has become possible because of the
availability of gene databases and the improved
understanding of metabolic processes. Based on
a combination of physiological data (traits),
genetic data (QTL, mutants), and gene databases,
a candidate gene is proposed. In the case of
mutants, the candidate gene, when mutated, is
expected to gives rise to the mutant phenotype.
In the case of QTL, there needs to be a tight
correlation between a specific allele and the
expression of the trait. It is sometimes hard
to come up with conclusive evidence in favor of
your candidate gene. The ultimate proof is when
you find that a mutant phenotype is restored to
wild type after introduction of an active copy of
the candidate gene (via transformation) (remember
T-DNA tagging!!). The second best scenario is
when you can show that independent mutant alleles
of the same gene all affect the same trait and
have similar phenotypes (remember
transposon-tagging!!). The least you can do is
show a correlation between the expression level
(at the RNA or protein level) of the candidate
gene and the trait of interest.
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Reverse genetics
Traditionally we are dealing with forward
genetics. In that case we have a mutant
phenotype and we like to know what gene is
mutated. Based on the identity of the gene and
the mutant genotype we can then infer the
function of that gene, or at the very least what
process the gene is involved in. An example is
the REF8 gene of Arabidopsis. The ref8 mutant,
which has the mutated copy of the REF8 gene
(Franke et al., 2002), accumulates a particular
type of lignin precursor, the H-unit. We can
therefore infer that the FUNCTIONAL REF8 gene is
involved in preventing the accumulation of
H-units. Reverse genetics deals with the
situation where we have a gene sequence, but we
do not know the function of the gene. In that
case we try to identify mutations in the gene of
interest, and evaluate the plants that carry the
mutations. Based on the mutant phenotype we can
then infer a function of the gene. This approach
tends to work best with mutations as a result of
T-DNA and transposon insertions, combined with
PCR.
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F2 population with active transposons
Pollinate (get seed)
Extract DNA
Pool DNA
Perform PCR on DNA pools with transposon primer
and GSP
DNA
Perform PCR on individuals of pools
Identify individual plant with insertion
Find seed observe plants
Will result in PCR product